Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha.
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII conti...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2024-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0285651&type=printable |
| _version_ | 1797315921983832064 |
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| author | Scott C Bolton David H Thompson Tamara L Kinzer-Ursem |
| author_facet | Scott C Bolton David H Thompson Tamara L Kinzer-Ursem |
| author_sort | Scott C Bolton |
| collection | DOAJ |
| description | Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKIIα holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versus Escherichia coli expression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII. |
| first_indexed | 2024-03-08T03:10:53Z |
| format | Article |
| id | doaj.art-7cee40b4adb741d7ae522cb3ea5e0c78 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-03-08T03:10:53Z |
| publishDate | 2024-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-7cee40b4adb741d7ae522cb3ea5e0c782024-02-13T05:34:05ZengPublic Library of Science (PLoS)PLoS ONE1932-62032024-01-01191e028565110.1371/journal.pone.0285651Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha.Scott C BoltonDavid H ThompsonTamara L Kinzer-UrsemCalcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKIIα holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versus Escherichia coli expression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0285651&type=printable |
| spellingShingle | Scott C Bolton David H Thompson Tamara L Kinzer-Ursem Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha. PLoS ONE |
| title | Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha. |
| title_full | Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha. |
| title_fullStr | Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha. |
| title_full_unstemmed | Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha. |
| title_short | Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha. |
| title_sort | methods optimization for the expression and purification of human calcium calmodulin dependent protein kinase ii alpha |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0285651&type=printable |
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